1. Field of the Art
n relates to oligonucleotide derivatives having amino groups introduced through intermediary straight or branched alkylene groups into the 5'-phosphate groups of oligonucleotide of a certain length, to an immobilized oligonucleotide bound to a carrier at the amino group moiety, and to a method for production of them.
In the field of biochemistry, purification of vital polymers is one of the important tasks of research, and a great deal of effort by a large number of researchers have been made in the past therefor. For this object, affinity chromatography techniques and electrophoresis using primarily polyacrylamide gel have been developed and appreciably utilized.
Many vital polymers have inherent properties to bind or interact specifically with specific substances. Affinity chromatography may be said to be a method utilizing skillfully the principle of biological discrimination possessed by vital polymers.
Today, when the affinity technique is undergoing rapid progress, it is being widely utilized for purification and separation of various vital substances, including, as a matter of course, proteins, enzymes, and also lipids, hormones, vitamins, and receptors.
Above all, affinity chromatography with the use of a nucleic acid as ligand is expected to be widely utilized in the future in various applications, including isolation of nucleic acids or proteins which are also important in molecular biology. Also, for the purpose of efficient isolation, it is of great interest to develop a crosslinking method between ligands and carriers.
2. Prior Art
From such a point of view, among the affinity chromatographies using carriers having nucleic acids bound thereto, the most widely utilized is the method in which RNA containing poly (A) at the 3'-end is isolated by the use of an oligo (dT)-cellulose or a poly (U)-agarose column [Ono, M., Kondo, T., Kawakami, M : J. Biochem., 81, 941 (1977)].
Poly (U), Poly (dA)-cellulose, etc. are used in the method wherein the base moiety of nucleotide is bound to a carrier activated with BrCN, etc., and therefore the resultant bound product is stable due to binding formed at multiple sites therebetween, while, on the other hand, it involves a drawback in that its adsorption capacity is weakened, because the base moieties necessary for affinity activity are used for binding with the carrier [Lindberg, U., Persson, T. : Eur. J. Biochem., 31, 246 (1972)].
Also, in the case of oligo (dT)-cellulose, binding between the hydroxyl groups of a carrier and the phosphoric acid groups of an oligonucleotide is said to be accomplished by the use of, for example, DCC (dicyclohexylcarbodiimide), but it involves the problems of non-specific adsorption and lack of reproducibility of adsorption capacity.
Other than the proposals of immobilized homopolymers of nucleotide as described above, there are several proposals in which DNA obtained from natural resources is immobilized [Anderson, J. N., Monahan J. J., O'Malley, B. W.: J. Biol. Chem., 252, 5789 (1977)], but there has, insofar as we are aware, been no report in the past that an oligonucleotide of a certain length having any desired base sequence has been bound to a carrier only at a specific position to be successfully immobilized.
Under these circumstances, if an oligonucleotide having any desired base sequence could be bound to a carrier at a specific site, such a technique would be useful not only for isolation and purification of a mRNA according to affinity chromatography utilizing the immobilized nucleotide homopolymer but also for isolation and purification of a mRNA having a specific base sequence. Further, its applicability for purificarion of various nucleic acid related enzymes recognizing specific base sequences may also be considered.
A large number of researches have also been carried out on affinity carriers by using mono- or di-nucleotides as ligand, and the results of some of them are now commercially available. However, the sites at which the nucleotide is bound to the carrier through an intermediary spacer are mostly the base moieties thereof.sup.(a). There are also some products in which the nucleotide is bound to the sites other than base moieties.sup.(b), but to the best of the present inventors' knowledge, such products involve the drawbacks of a large number of steps required for synthesis of the starting ligand and cumbersome procedures over the entire synthesis. Also, none of the methods can be used for oligonucleotide.
(a) Lee, C. Y., Lappi, D. A., Wermuth, B., Everse, J., Kaplan, N. O.: Arch. Biochem. Biophs., 168, 561 (1974); Ishiwata, K., Yoshida, H. : J. Biochem., 83, 783 (1978); Japanese Patent Laid-Open Nos. 25795/1977, 101396/1978, 133283/1978 and 36277/1980.
(b) Jervis, L., Pettit, N. M.: J. Chromatog., 97, 33 (1974); Lamed, R., Levin, Y., Wilchek, M. : Biochem. Biophys. Acta., 304, 231 (1973); Janski, A., Oleson, A. E. : Anal. Biochem., 71, 471 (1976).